Fig 1: Combined application of CXCR2 inhibitor SB225002 and anti-PD-L1 dramatically blocks HOXC10-mediated CRC metastasis. (A-E). One week after injection of MC38-HOXC10 cells, mice in four groups were treated with vehicle, PD-L1 antibody or/and SB225002 (n=10 mice/group) until treatment endpoint. In vivo assays showed that combined treatment of PD-L1 antibody and CXCR2 inhibitors SB225002 can almost inhibit CRC metastasis. (A). Representative bioluminescent images and bioluminescent signals in indicated group. (B). Overall survival in indicated group. (C). The number of lung and liver nodules in indicated group. (D). The incidence of lung and liver nodules in indicated group. (E). Representative HE staining of lung and liver tissues. (F). IHC staining detected the infiltration of MDSCs or CD8+ cells in indicated group. All the data are shown as the mean±s.d. * P<0.05.
Fig 2: Clinical relevance of HOXC10 and VEGFA expression in gliomas. (A) HOXC10 levels were positively associated with VEGFA expression in 94 primary human glioma specimens. Left panel: two representative cases are shown. Magnification ×400: scale bars, 50 μm (top 2 rows). Magnification ×1,000: scale bars, 25 μm (bottom 2 rows). Right panel: percentage of glioma specimens showing low or high HOXC10 expression relative to the level of VEGFA. (B) Expression analysis (left) and correlation (right) between HOXC10 expression and VEGFA expression in 10 freshly collected human glioma samples. The western blotting assay was replicated independently three times.
Fig 3: HOXC10 interacts with PRMT5 to promote VEGFA transcription. (A) ChIP-qPCR enrichments of histone post-translational modifications on VEGFA promoter region 3 in the indicated cells, displayed using a heatmap. The color key represents an intensity scale for ratio of HOXC10 versus vector or HOXC10-shRNA versus shRNA control vector, normalized by log2 transformation. (B) ChIP-qPCR enrichments of PRMT5 or lgG on VEGFA promoter region 3 in the indicated cells. (C) ChIP-qPCR enrichments of HOXC10 or lgG on VEGFA promoter region 3 in the control-shRNA-transduced, HOXC10-overexpressing glioma cells or PRMT5-shRNA-transduced, HOXC10-overexpressing glioma cells. (D) Vector or Flag-tagged HOXC10 was transfected into the U251MG or LN229. Co-IP assay showed that HOXC10 interacted with PRMT5. (E) Co-IP assay showed that endogenous HOXC10 interacted with PRMT5 in U251MG and LN229 cells. (F) Far western blotting analysis showed that Flag-tagged HOXC10 in U251MG transfected with Flag-tagged HOXC10 interacted with recombinant GST-PRMT5. (G) Schematic illustration of full length HOXC10 or HOXC10 truncations. (H) Flag-tagged HOXC10 (full length or truncations) and HA-tagged PRMT5 were transfected into U251MG. Co-IP assay revealed that PRMT5 interacted with FL, F1 and F4, but not with F2 and F3. (I) VEGFA promoter luciferase reporter plasmids, Renilla pRL-TK plasmids, vector, or Flag-tagged HOXC10 (full length or truncations) were transfected into U251MG cells. After 48 h, cells were subjected to a luciferase reporter assay. All the assays shown in the figure were independently replicated three times. Error bars, SD. * P < 0.05.
Fig 4: HOXC10 accelerates OC cell migration. (A, B) Relative mRNA and protein expression levels of HOXC10. 8910 vs. PM cells. P=0.0177 (mRNA), P=0.0301 (protein). (C) Comparison of the migration ability of 8910 and PM cells via wound healing assays. P=0.0002. Scale bars, 200 μm. (D, E) Transfection efficiencies of the HOXC10 siRNA products. P<0.0001, P=0.0001 and P<0.0001; P=0.0008, P=0.0120 and P=0.0013. (F) Transwell assay of 8910 and PM cells transfected with HOXC10 siRNA products No. 1 and No. 3 and the negative control siRNA. 8910 cell graphs, P<0.0001 and P<0.0001. PM cell graphs, P<0.0001 and P<0.0001. Scale bars, 100 μm. (G, H) Wound healing assay of 8910 and PM cells transfected with HOXC10 siRNA products No. 1 and No. 3 and the negative control siRNA. P=0.0010 and P=0.0167; P=0.0025 and P=0.0097. Scale bars, 200 μm.
Fig 5: Overexpression of HOXC10 is associated with poor prognosis in OC patients. (A) Kaplan-Meier survival curves for OC patients (PFS; n = 1207). HR=1.17, P=0.031. (B) HOXC10 protein expression in normal ovarian tissues (case 1) and OC patient tissues (case 2, low expression of HOXC10; case 3, high expression of HOXC10) was assessed by IHC staining. Scale bars, 50 μm. (C) IHC positive rate scores for HOXC10 in normal tissues and each group of OC patient tissues. P<0.0001. (D) Kaplan-Meier survival curves for 158 OC patients. HR=1.754, P=0.0096.
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